Tyrosine Sulfation Influences the Chemokine Binding Selectivity of Peptides Derived from Chemokine Receptor CCR3

Abstract

The interactions of chemokines with their G protein-coupled receptors play critical roles in the control of leukocyte trafficking in normal homeostasis and in inflammatory responses. Tyrosine sulfation is a common post-translational modification in the amino-terminal regions of chemokine receptors. However, tyrosine sulfation of chemokine receptors is commonly incomplete or heterogeneous. To investigate the possibility that differential sulfation of two adjacent tyrosine residues could bias the responses of chemokine receptor CCR3 to different chemokines, we have studied the binding of three chemokines (eotaxin-1/CCL11, eotaxin-2/CCL24, and eotaxin-3/CCL26) to an N-terminal CCR3-derived peptide in each of its four possible sulfation states. Whereas the nonsulfated peptide binds to the three chemokines with approximately equal affinity, sulfation of Tyr-16 gives rise to 9-16-fold selectivity for eotaxin-1 over the other two chemokines. Subsequent sulfation of Tyr-17 contributes additively to the affinity for eotaxin-1 and eotaxin-2 but cooperatively to the affinity for eotaxin-3. The doubly sulfated peptide selectively binds to both eotaxin-1 and eotaxin-3 approximately 10-fold more tightly than to eotaxin-2. Nuclear magnetic resonance chemical shift mapping indicates that these variations in affinity probably result from only subtle differences in the chemokine surfaces interacting with these receptor peptides. These data support the proposal that variations in sulfation states or levels may regulate the responsiveness of chemokine receptors to their cognate chemokines.

Design and Receptor Interactions of Obligate Dimeric Mutant of Chemokine Monocyte Chemoattractant Protein-1 (MCP-1)

“…S3). These are the same regions that are sensitive to peptide binding in MCP-1(P8A) and also correspond to the chemokine regions found to interact with receptor peptides in previous studies of other chemokines (15,25,(27)(28)(29)(30)(31)(32)(33). Thus, it appears likely that the observed interactions represent a conserved aspect of chemokine-receptor recognition and could occur in the context of the full-length receptor.…”

Tyrosine Sulfation of Chemokine Receptor CCR2 Enhances Interactions with Both Monomeric and Dimeric Forms of the Chemokine Monocyte Chemoattractant Protein-1 (MCP-1)

“…5) Second, the residues of MCP-1(P8A) generally had higher ⌬␦ NH values than the residues of MCP-1(T10C), particularly for binding to R2B and R2D. As we have noted in previous sulfopeptide binding studies (13,15), larger chemical shift changes generally occur for tighter binding protein-peptide pairs. Third, in many cases the same regions of the two MCP-1 mutants displayed chemical shift changes upon binding to the peptides; these regions included the N-loop (residues 13-21), the 3 10 turn (residues 22-24), the 40s loop (residues 46 -49), and the third ␤-sheet (residues 50 -54).…”

“…Nevertheless, extensive mutational analysis has identified the residues on MCP-1 that are involved in receptor binding and activation (20,31). Critical residues for binding to CCR2 were found to be: Tyr 13 , Arg MCP-1 for the N-terminal region of CCR2. Fig.…”

“…Thus, the current NMR data are consistent with the previous mutational data and indicate that the likely binding site for the receptor N terminus is a shallow cleft between the N-loop and ␤3-strand on the chemokine. Previous NMR studies of other chemokines have also implicated the N-loop and ␤3-strand regions in recognition of the N-terminal regions of chemokine receptors (13)(14)(15)(32)(33)(34)(35)(36)(37). In contrast, the 30s loop residues of MCP-1 only showed minor changes of chemical shift, but mutations in this region decreased CCR2 binding (20).…”

“…The chemokine receptors CCR2, CCR5, CCR8, CXCR3, CXCR4, CX 3 CR1, and Duffy antigen and receptor for chemokines (DARC) have been demonstrated to contain sulfated tyrosine residues that modulate chemokine binding (5)(6)(7)(8)(9)(10)(11). Moreover, studies using differentially sulfated N-terminal peptides from various chemokine receptors have shown that tyrosine sulfation increases the binding affinity of the receptor peptides to their cognate chemokines (12)(13)(14)(15)(16).…”